3e7j | pdb_00003e7j
HeparinaseII H202A/Y257A double mutant complexed with a heparan sulfate tetrasaccharide substrate
Structural highlights
Function[HEPB_PEDHD] Cleaves both heparin and heparan sulfate glycosaminoglycans through a beta-elimination mechanism. Cleaves heparin at alpha-D-GlcNp2S6S(1->4) alpha-L-IdoAp2S and heparan sulfate at alpha-D-GlcNp2Ac(or 2S)6OH(1->4)beta-D-GlcAp.[1] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedHeparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a beta-elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles. Catalytic mechanism of heparinase II investigated by site-directed mutagenesis and the crystal structure with its substrate.,Shaya D, Zhao W, Garron ML, Xiao Z, Cui Q, Zhang Z, Sulea T, Linhardt RJ, Cygler M J Biol Chem. 2010 Jun 25;285(26):20051-61. Epub 2010 Apr 19. PMID:20404324[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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